Polypeptide variants

ABSTRACT

The invention relates to the provision of oligomeric polypeptides (dimers, trimmers, etc) comprising the ligand binding domains of cytokines which are linked via flexible polypeptides linker molecules. The linker molecules optionally comprise protease sensitive sites to modulate the release of biologically active cytokines when administered to a human or animal subject.

The invention relates to polypeptides which comprise more than twoligand binding domains of a cytokine wherein the domains are linked by aflexible linker which optionally comprises a proteolytic cleavage site.

Ligands which interact with receptors to bring about a suitablebiochemical response are known as agonists and those that prevent, orhinder, a biochemical response are known as antagonists. For example,and not by way of limitation, cell specific growth factors are ligandsthat act as agonists and bind receptors located at the cell surface.Activation of the receptors by ligand-specific binding promotes cellproliferation via activation of intracellular signalling cascades thatresult in the expression of, amongst other things, cell-cycle specificgenes, and the activation of quiescent cells to proliferate. Growthfactors may also activate cellular differentiation.

A large group of growth factors, referred to as cytokines, are involvedin a number of diverse cellular functions. These include, by example andnot by way of limitation, modulation of the immune system, regulation ofenergy metabolism and control of growth and development. Cytokines whichare secreted by lymphocytes are termed lymphokines (also known asinterleukins). Those secreted by monocytes and macrophages are termedmonokines. Cytokines are also secreted by endocrine glands, (for examplegrowth hormone (GH) by the pituitary gland), and adipose cells (forexample leptin). Cytokines mediate their effects via receptors expressedat the cell surface of target cells.

Receptors of the cytokine receptor family possess a single transmembranedomain and lack intrinsic enzyme activity (Kishimoto et al., 1994). Uponbinding of a cytokine to a cognate receptor, either receptor homo- orhetero-dimerisation or oligomerisation occurs. The receptor complex isinternalised and signalling occurs through the activation of associatedsignalling cascades that include the Jak/Stat and Mapk pathways.Internalisation is followed by a recycling step whereby the receptormolecule is regenerated for further use within the cell.

An example of the above is described with respect to GH and its bindingto the GHR. This example is merely meant to be illustrative and notlimiting and is an example of a cytokine which activates a signaltransduction cascade by binding, dimerisation and internalisation of thereceptor:ligand complex. It is known that a single molecule of growthhormone (GH) associates with two identical receptor molecules(Cunningham et al., 1991; de Vos et al., 1992; Sundstrom et al., 1996;Clackson et al., 1998). This occurs through two unique receptor-bindingsites on GH and a common binding pocket on the extracellular domain oftwo receptors. Site 1 on the GH molecule has a higher affinity than site2, and receptor dimerization is thought to occur sequentially with onereceptor binding to site 1 on GH followed by recruitment of a secondreceptor to site 2.

The extracellular portion of the GHR exists as two linked domains eachof approximately 100 amino acids (SD-100), the C-terminal SD-100 domainbeing closest to the cell surface and the N-terminal SD-100 domain beingfurthest away. It is a conformational change in these two domains thatoccurs on hormone binding with the formation of the hetero-trimericcomplex GHR-GH-GHR. It has been proposed that ligand-driven receptordimerization is the key event leading to signal activation (Cunninghamet al., 1991), triggering phosphorylation cascades that include theJak2/Stat5 pathway (Argetsinger & Carter-Su, 1996). Using confocalmicroscopy and Fluorescence Resonance Energy Transfer (FRET) it is knownthat there is very rapid internalisation of GHR after ligand binding andthat internalisation and signaling are independent functions (Maamra etal., 1999). Internalisation of the GHR-GH-GHR complex is followed by arecycling step whereby the receptor molecule is regenerated for furtheruse within the cell.

Examples of cytokines which are related to GH are leptin anderythropoietin (EPO). The leptin receptor and the EPO receptor shareconsiderable structural homology with the GHR and require a similardimerisation process to trigger signalling. Leptin supresses appetiteand leptin resistance is associated with obesity. A leptin receptorantagonist will provide a treatment for anorexia nervosa. EPO excesscauses polycythaemia which may be secondary to hypoxia (chronic lungdisease), or primary in the case of polycythaemia rubra vera (a disorderof excess red blood cells). An EPO antagonist will provide a therapy forpolycythaemia.

The disorders of acromegaly and gigantism result from an excess ofgrowth hormone, usually due to pituitary tumours. A drug currently undertrial is the pegylated GH antagonist B2036, designed using recentlyacquired knowledge of the molecular structure of the GHR. Unfortunately,to antagonise GH action very high levels of B2036 are required, over a1000 times higher than endogenous GH levels.

The invention relates, inter alia, to the provision of oligomericpolypeptides (dimers, trimers etc) comprising the ligand binding domainsof cytokines which are linked via flexible polypeptide linker moleculeswhich optionally include protease sensitive sites to modulate therelease of biologically active cytokines when administered to an animal.

According to a first aspect of the invention there is provided apolypeptide comprising more than two ligand binding domains of acytokine receptor wherein said domains are linked by a linker molecule.

Preferably the linker molecule comprises at least one proteolyticcleavage site.

Preferably said cleavage site is sensitive to a serum protease.

Preferably said cleavage site comprises the amino acid sequence: LVPRGS(SEQ ID:1), or variant thereof.

In a further preferred embodiment of the invention said cleavage sitecomprises at least one copy of the amino acid sequence: SGGGG (SEQID:2), or functional variant thereof. Preferably, said cleavage sitecomprises the amino acid sequence PGISGGGGGG (SEQ ID:3).

More preferably still said cleavage site comprises the amino acidsequence: LVPRGSPGISGGGGGG (SEQ ID:4), or variant thereof.

Alternatively, said cleavage site comprises at least two copies of theamino acid sequence SGGGG, or functional variant thereof, which flanksaid cleavage site.

In a further preferred embodiment of the invention said cleavage site issensitive to the serum protease thrombin.

In a further preferred embodiment of the invention said polypeptidecomprises a plurality of ligand binding domains. Preferably saidpolypeptide has 3, 4, 5, 6, 7, 8, 9, or 10 ligand binding domains.Preferably said polypeptide has greater than 10 ligand binding domains.

In a further preferred embodiment of the invention said polypeptide has4, 6, 8, 10, or 12 ligand binding domains.

In a preferred embodiment of the invention said polypeptide comprises atleast four ligand binding domains.

In a further embodiment of the invention said polypeptide is anantagonist.

In an alternative preferred embodiment of the invention said polypeptideis an agonist.

In a further preferred embodiment of the invention said ligand bindingdomain is selected from the ligand binding domains of the cytokinesselected from the group consisting of: growth hormone; leptin;erythropoietin; prolactin; interleukins (IL), IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-9, IL-10, IL-11; the p35 subunit of IL-12, IL-13, IL-15;granulocyte colony stimulating factor (G-CSF); granulocyte macrophagecolony stimulating factor (GM-CSF); ciliary neurotrophic factor (CNTF);cardiotrophin-1 (CT-1); leukaemia inhibitory factor (LIF); oncostatin M(OSM); interferon, IFNα and IFNγ.

In a preferred embodiment of the invention said ligand binding domain isthe ligand binding domain of growth hormone.

A single hGH molecule binds to two GHR molecules. The hGH moleculeinteracts with one receptor molecule through a high affinity site, andwith the other through a low affinity site. A single protein chainconsisting of hGH linked to hGH will contain two high affinity siteswhich can bind strongly to a pair of receptor molecules and two lowaffinity sites.

In one embodiment of the invention, two or more copies of a ligandbinding domain are expressed on a single polypeptide chain and thesequence of the tandem or oligomeric cytokine is arranged ‘ligandbinding domain-linker-ligand binding domain’.

In a further preferred embodiment of the invention said ligand bindingdomain is the binding domain of leptin.

Preferably the linker comprises at least one copy of the peptide:

Gly Gly Gly Gly Ser Ser Ser Ser (hereinafter referred to as “Gly4Ser4”)(SEQ ID:5).

In one embodiment of the invention the linker is 8 amino acids in lengthand consists of one copy of the Gly4Ser4 linker. In an alternativeembodiment of the invention, the linker is 16 amino acids in length andconsists of two copies of the Gly4Ser4 linker. In a further alternativeembodiment of the invention said linker is 24 amino acids in length andconsists of three copies of the Gly4Ser4 linker.

More preferably the linker is a polypeptide which comprises 5 to 50amino acid residues. Most preferably the linker comprises 5 to 30 aminoacid residues.

Most preferably the linker comprises at least one copy of the peptide:

Gly Gly Gly Gly Ser (hereinafter referred to as “(Gly4Ser)” or (G₄S))(SEQ ID:6).

In one embodiment of the invention the linker is 5 amino acids in lengthand consists of one copy of the (Gly4Ser) linker. In an alternativeembodiment of the invention, the linker is 10 amino acids in length andconsists of two copies of the (Gly4Ser)2 linker. In a furtheralternative embodiment of the invention said linker is 15 amino acids inlength and consists of three copies of the (Gly4Ser) linker. In afurther alternative embodiment of the invention said linker is 20 aminoacids in length and consists of 4 copies of the (Gly4Ser)4 linker.

In an alternative embodiment of the invention, the polypeptide is afusion protein comprising inframe translational fusions of ligandbinding domains according to the invention.

It will be apparent to one skilled in the art that alternative linkerscan be used to link ligand binding domains, for example the use ofchemical protein crosslinkers. For example homo-bifunctional crosslinkersuch as disuccinimidyl-suberimidate-dihydrochloride;dimethyl-adipimidate-dihydrochloride; 1,5,-2,4 dinitrobenezene orhetero-bifunctional crosslinkers such as N-hydroxysuccinimidyl 2,3-dibromopropionate; 1-ethyl-3-[3-dimethylaminopropyl] carbodiimidehydrochloride; succinimidyl4-[n-maleimidomethyl]-cyclohexane-1-carboxylate.

Further examples of chemical crosslinks include the provision ofchemically modified linker molecules and/or ligand binding domains. Forexample, if one end of the linker molecule is terminated with an aminoterminal lysine residue and the ligand binding with a carboxyl-terminalglutamine residue then ligand binding domains can be oligomerised withtransglutaminase.

According to a further aspect of the invention there is provided anucleic acid molecule comprising a nucleic acid sequence which encodes apolypeptide according to the invention.

In a preferred embodiment of the invention said nucleic acid moleculecomprises a nucleic acid sequence selected from the group consisting of:

i) a sequence represented by FIGS. 4 or 6;

ii) a sequence which hybridises to the sequence of (i) above and whichhas cytokine receptor modulating activity; and

iii) a sequence which is degenerate as a result of the genetic code tothe sequences defined in (i) and (ii) above.

In a preferred embodiment of the invention said nucleic acid hybridisesunder stringent hybridisation conditions to the sequences represented inFIGS. 4 or 6.

It is well known in the art that optimal hybridisation conditions can becalculated if the sequence of the nucleic acid is known. For example,hybridisation conditions can be determined by the GC content of thenucleic acid subject to hybridisation. Please see Sambrook et al. (1989)Molecular Cloning; A Laboratory Approach. A common formula forcalculating the stringency conditions required to achieve hybridisationbetween nucleic acid molecules of a specified homology is:T_(m)=81.5° C.+16.6 Log[Na⁺]+0.41[% G+C]−0.63 (% formamide).

Typically, hybridisation conditions uses 4-6×SSPE (20×SSPE contains175.3 g NaCl, 88.2 g NaH₂PO₄H₂O and 7.4 g EDTA dissolved to 1 litre andthe pH adjusted to 7.4); 5-10× Denhardts solution (50× Denhardtssolution contains 5 g Ficoll (type 400, Pharmacia), 5 gpolyvinylpyrrolidone abd 5 g bovine serum albumen/500 ml; 100 μg-1.0mg/ml sonicated salmon/herring DNA; 0.1-1.0% sodium dodecyl sulphate;optionally 40-60% deionised formamide. Hybridisation temperature willvary depending on the GC content of the nucleic acid target sequence butwill typically be between 42°-65° C.

According to a further aspect of the invention there is provided apolypeptide which is encoded by a nucleic acid molecule according to theinvention.

In a preferred embodiment of the invention the polypeptide so encoded ismodified by deletion, addition or substitution of at least one aminoacid residue. Ideally said modification enhances the antagonistic oragonistic effects of said polypeptide with respect to the inhibition oractivation of receptor mediated cell signalling.

Alternatively, or preferably, said modification includes the use ofmodified amino acids in the production of recombinant or synthetic formsof polypeptides.

It will be apparent to one skilled in the art that modified amino acidsinclude, by way of example and not by way of limitation,4-hydroxyproline, 5-hydroxylysine, N⁶-acetyllysine, N⁶-methyllysine,N⁶,N⁶-dimethyllysine, N⁶,N⁶,N6-trimethyllysine, cyclohexyalanine,D-amino acids, ornithine. The incorporation of modified amino acids mayconfer advantageous properties on polypeptides. For example, theincorporation of modified amino acids may increase the affinity of thepolypeptide for its binding site, or the modified amino acids may conferincreased in vivo stability on the polypeptide thus allowing a decreasein the effective amount of therapeutic polypeptide administered to apatient.

According to a yet further aspect of the invention there is provided avector including a DNA molecule according to any preceding aspect orembodiment of the invention.

In a preferred embodiment of the invention said vector is provided withmeans to manufacture recombinantly the polypeptide of the invention.

In a preferred embodiment of the invention said vector is an expressionvector adapted for prokaryotic gene expression.

Prokaryotic expression systems are well known in the art and comprisevectors adapted for high level constitutive and inducible expression.Inducible expression systems are particularly advantageous if therecombinant polypeptide is toxic to the bacterial cell. Induction ofexpression is tightly regulated by promoters responsive to variousinducers (e.g. IPTG inducible). Bacterial cells can be grown tostationary phase before induction thereby reducing harmful effects oftoxic polypeptides.

Additionally it is also well known in the art that certain polypeptidesare difficult to manufacture recombinantly due, for example, to proteininstability or problems of aggregation. It is well known thatgenetically modified bacterial strains are available which are mutatedin genes (e.g. bacterial proteases) which facilitate the production ofnative and recombinant bacterial polypeptides.

In a further preferred embodiment of the invention said vector is anexpression vector adapted for eukaryotic gene expression.

Typically said adaptation includes, by example and not by way oflimitation, the provision of transcription control sequences (promotersequences) which mediate cell/tissue specific expression. These promotersequences may be cell/tissue specific, inducible or constitutive.

Promoter is an art recognised term and, for the sake of clarity,includes the following features which are provided by example only, andnot by way of limitation. Enhancer elements are cis acting nucleic acidsequences often found 5′ to the transcription initiation site of a gene(enhancers can also be found 3′ to a gene sequence or even located inintronic sequences and are therefore position independent). Enhancersfunction to increase the rate of transcription of the gene to which theenhancer is linked. Enhancer activity is responsive to trans actingtranscription factors (polypeptides) which have been shown to bindspecifically to enhancer elements. The binding/activity of transcriptionfactors (please see Eukaryotic Transcription Factors, by David S.Latchman, Academic Press Ltd., San Diego) is responsive to a number ofenvironmental cues which include, by example and not by way oflimitation, intermediary metabolites (eg glucose, lipids), environmentaleffectors (eg light, heat).

Promoter elements also include so called TATA box and RNA polymeraseinitiation selection (RIS) sequences which function to select a site oftranscription initiation. These sequences also bind polypeptides whichfunction, inter alia, to facilitate transcription initiation selectionby RNA polymerase.

Adaptations also include the provision of selectable markers andautonomous replication sequences which both facilitate the maintenanceof said vector in either the eukaryotic cell or prokaryotic host.Vectors which are maintained autonomously are referred to as episomalvectors.

Adaptations which facilitate the expression of vector encoded genesinclude the provision of transcription termination/polyadenylationsequences. This also includes the provision of internal ribosome entrysites (IRES) which function to maximise expression of vector encodedgenes arranged in bicistronic or multi-cistronic expression cassettes.

These adaptations are well known in the art. There is a significantamount of published literature with respect to expression vectorconstruction and recombinant DNA techniques in general. Please see,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory, Cold Spring Harbour, N.Y. and referencestherein; Marston, F (1987) DNA Cloning Techniques: A Practical ApproachVol. III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

In yet a further aspect of the invention there is provided a method toprepare a polypeptide according to the invention comprising:

(i) growing a cell transformed or transfected with a nucleic acid orvector of the present invention in conditions conducive to themanufacture of said polypeptide; and

(ii) purifying said polypeptide from said cell, or its growthenvironment.

In a preferred method of the invention said vector encodes, and thussaid recombinant polypeptide is provided with, a secretion signal tofacilitate purification of said binding agent polypeptide.

In yet a further aspect of the invention there is provided a celltransformed/transfected with the vector or nucleic acid according to theinvention.

Preferably said cell eukaryotic and is selected from: fungal; insect(e.g. Spodoptera frugiperda); amphibian; plant; mammalian.

More preferably said cell is prokaryotic and is an E. coli cell.

According to a further aspect of the invention there is provided the useof the polypeptide according to the invention as a pharmaceutical.Preferably there is provided a pharmaceutical composition comprising thepolypeptide according to the invention. Preferably said pharmaceuticalcomposition includes a carrier, excipient and/or a diluent.

In a further preferred embodiment of the present invention saidpolypeptide is used for the manufacture of a medicament for use in thetreatment of a disease selected from the group consisting of:acromegaly; gigantism; GH deficiency; Turners syndrome; renal failure;osteoporosis; diabetes mellitus; cancer; obesity; insulin resistance;hyperlipidaemia; hypertension; anaemia; autoimmune and infectiousdisease; inflammatory disorders including rheumatoid arthritis.

The invention also provides for a method of treating a human or animalsubject comprising administering an effective amount of the polypeptide,pharmaceutical composition or medicament to said subject.

The polypeptides or compositions of the invention can be administered byany conventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, transdermalor delivered by a non-pathological GMO engineered to secrete thepolypeptide.

The pharmaceutical compositions used in the foregoing methods preferablyare sterile and contain an effective amount of the polypeptide accordingto the invention for producing the desired response in a unit of weightor volume suitable for administration to a patient.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents (e.g., aceticacid in a salt; citric acid in a salt; boric acid in a salt; andphosphoric acid in a salt), preservatives (e.g., benzalkonium chloride;chlorobutanol; parabens and thimerosal, compatible carriers, andoptionally other therapeutic agents.

Compositions may be combined, if desired, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation of polypeptideswhich is preferably isotonic with the blood of the recipient. Thispreparation may be formulated according to known methods using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation also may be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Carrier formulation suitable fororal, subcutaneous, intravenous, intramuscular, etc. administrations canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

According to a further aspect of the invention there is provide a methodof treatment comprising administering to an animal, preferably a human,an effective amount of the nucleic acid or vector according to theinvention.

An embodiment of the invention will now be described by example only andwith reference to the following figures wherein;

FIG. 1 illustrates a plasmid map of pTrcHisχ1A1.

FIG. 2 illustrates primers used in the synthesis of the tandemconstructs.

FIG. 3 is a schematic diagram for the construction of pTrcHisχ1C1.pTrcHisχ1C2 is constructed in the same way, however the forward primerused is DiGHNotF and the restriction enzymes used are NotI and HindIII.

FIG. 4 illustrates the DNA sequence for growth hormone tandem segregatedby a thrombin cleavable linker. The linker region is shown in italics,with the thrombin cleavage site in bold.

FIG. 5 illustrates the protein sequence for growth hormone tandemsegregated by a thrombin cleavable linker. The linker region is shown initalics, with the thrombin cleavage site in bold.

FIG. 6 illustrates the DNA sequence for leptin tandem segregated by athrombin cleavable linker. The linker region is shown in italics, withthe thrombin cleavage site in bold.

FIG. 7 illustrates the protein sequence for leptin tandem segregated bya thrombin cleavable linker. The linker region is shown in italics, withthe thrombin cleavage site in bold. The cleavable linker is optional inthis construct.

FIG. 8 shows the Coomassie stained SDS-PAGE gel and western blot of thepurified GH-tandem (χ1C1).

FIG. 9 is the bioassay data generated for χ1C1, with data for in-housesynthesised GH and commercially produced GH for comparison. The activityof the tandem is similar to the in-house generated GH. In each case theconcentrations of the test protein used were 0 ng, 6.25 ng, 12.5 ng, 25ng, 50 ng and 100 ng.

FIG. 10 is a schematic diagram illustrating the construction ofpTrcHisχ2C1.

FIG. 11 shows the western blot of the SDS-PAGE gel on which the proteinsexpressed by clones of E. coli SURE:pTrcHisχ2C1 cells were run. Theexpected size of the leptin-tandem is ˜37 kDa and a faint band isobserved at this size. The major band however is approximately half ofthis size. This suggests that the leptin tandem is being expressed butis most probably cleaved to the single leptin domains.

MATERIALS AND METHODS

The sequence encoding GHR in pTrcHisχ1A1 (FIG. 1) was replaced withanother GH gene (residues 1-191) to produce two successive GH geneslinked with a (G₄S)₄ linker. The resultant construct (pTrcHisχ1C1) wastransformed into E. coli SURE cells, a DNA recombination deficientstrain of E. coli. Clones expressing GH-(G₄S)₄-GH protein wereidentified by western blotting using anti-GH (10A7, mouse IgG1) probedwith Sheep anti-mouse-HRP (Amersham). Another GH-tandem, which lackedthe (G₄S)₄ linker (GH-GH) was also constructed (pTrcHisχ1C2) using thesame method.

GH-tandem protein was purified from cell lysates using a metal chelateaffinity column (Probond resin, Invitrogen) followed by an ion exchangecolumn (MonoQ, Pharmacia).

The effect of the GH-tandem proteins were analysed using an establishedbioassay (Ross et al., 1997).

The leptin gene was originally cloned into pHEAT; atemperature-inducible vector. However for expression in E. coli SUREcells the gene was sub-cloned into the pTrcHis plasmid. A (G₄S)₄ linkerwas then introduced and finally the second leptin domain was ligatedinto the gene to produce the construct that would express the leptintandem. This construct pTrcHisχ2C1 was then transformed into E. coliSURE cells. Expression of the leptin-tandem was verified by western blotusing anti-leptin antibodies (Sigma), developed in rabbit, probed withanti-rabbit-HRP (Sigma).

Cloning of the GH-Tandems

PCR was used to produce a fragment of DNA which consisted of arestriction site (NotI or EcoRI), followed by the GH gene and then aHindIII restriction site. This was ligated into pTrcHisχ1A1, which hadbeen digested with the relevant restriction enzymes, to produce theconstructs χ1C1 (GH-(G₄S)₄-GH) and χ1C2 (GH-GH). The primers used forthe PCR's are shown in FIG. 2, and the reaction scheme is shown in FIG.3.

Purification of the GH-Tandems

Induced cells (resuspended in 20 mM sodium phosphate buffer, 500 mMsodium chloride, pH 7.8) were lysed with a combination of 100 μg/ml(final concentration) hen egg white lysozyme and sonication. Insolublematerial was removed by centrifugation at 400 rpm for 20 minutes.

The cleared cell lysate was applied to a 5 ml Probond resin column(Invitrogen), equilibrated with 20 mM sodium phosphate buffer, 500 mMsodium chloride, 5% glycerol, pH 7.8. The column was then washed with 10column volumes of 20 mM sodium phosphate buffer, 500 mM sodium chloride,5% glycerol, pH 6.0. Bound protein was eluted using 5 ml 20 mM sodiumphosphate buffer, 500 mM sodium chloride, 5% glycerol, 500 mM imidazole,pH 6.0.

The protein was dialysed overnight against Low Salt Buffer (25 mM TRIS,1 mM EDTA, 5% glycerol, pH 8.0) and then centrifuged to remove anyparticulate matter.

The protein sample was then loaded onto a Mono-Q column (Pharmacia),which had been pre-equilibrated with Low Salt Buffer. After a 10 columnvolume wash with Low Salt Buffer, the bound proteins were eluted over 20column volumes using a gradient between 0M sodium chloride to 1M sodiumchloride (in 25 mM TRIS, 1 mM EDTA, 5% glycerol, pH8.0). Peaks on theelution profile were analysed by SDS-PAGE and western blotting.

GH-tandem protein was then concentrated (if required) using a AmiconCentriprep Y-10 column.

The purity of the purified χ1C1 was confirmed by SDS-PAGE, by bothcoomassie staining and western blot (FIG. 8). Once the integrity of thissample had been confirmed, χ1C1 was submitted to the previouslyestablished bioassay (Ross et al., 1997) (FIG. 9).

Cloning of the Leptin-Tandems

PCR using the primers Lep2TrcFOR and Lep2TrcREV (FIG. 2) was used togenerate DNA sequence consisting of(NheI)-LEPTIN-(NotI)-(XhoI)-(SalI)-STOP-(EcoRI) from pHEATLeptin. Theterminal restriction sites were introduced by PCR and the internalrestriction sites were already present in the pHEATLeptin vector. ThePCR product generated was ligated into pTrcHisχ1A1 between NheI andEcoRI restriction sites to produce pTrcHisLeptin.

PCR was then used to generate the (G4S)₄-encoding linker flanked by NotIand XhoI restriction sites, the primers used were LepLinkFOR andLepLinkREV (FIG. 2). This was ligated into pTrcHisLeptin between theNotI and XhoI sites to produce pTrcHisLepLink.

The second leptin gene flanked by XhoI and SalI restriction sites wasgenerated by PCR, using the primers Lep2FOR and Lep2REV (FIG. 2). Thiswas ligated between the XhoI and SalI restriction sites to produce theconstruct which would express the leptin tandem, pTrcHisχ2C 1. Thisprocess for the generation of the leptin-tandem is shown in FIG. 10.

This plasmid was transformed into E. coli SURE cells and expressionstudies carried out, visualisation of expression was performed bywestern blot (FIG. 11)

References

-   ARGETSINGER, L. S. & CARTER-SU, C. (1996) Growth hormone signalling    mechanisms: involvement of the tyrosine kinase JAK2. [Review] [19    refs]. Hormone Research, 45 Suppl 1, 22-24.-   CHEN, C., BRINKWORTH, R. & WATERS, M. J. (1997) The role of receptor    dimerization domain residues in growth hormone signaling. Journal of    Biological Chemistry, 272, 5133-5140.-   CHEN, W. Y., CHEN, N. Y., YUN, J., WAGNER, T. E. &    KOPCHICK, J. J. (1994) In vitro and in vivo studies of antagonistic    effects of human growth hormone analogs [published erratum appears    in J Biol Chem 1994 Aug. 12;269(32):20806]. Journal of Biological    Chemistry, 269, 15892-15897.-   CHEN, W. Y., WHITE, M. E., WAGNER, T. E. & KOPCHICK, J. J. (1991)    Functional antagonism between endogenous mouse growth hormone (GH)    and a GH analog results in dwarf transgenic mice. Endocrinology,    129, 1402-1408.-   CHEN, W. Y., WIGHT, D. C., MEHTA, B. V., WAGNER, T. E. &    KOPCHICK, J. J. (1991) Glycine 119 of bovine growth hormone is    critical for growth-promoting activity. Molecular Endocrinology, 5,    1845-1852.-   CHEN, W. Y., WIGHT, D. C., WAGNER, T. E. & KOPCHICK, J. J. (1990)    Expression of a mutated bovine growth hormone gene suppresses growth    of transgenic mice. Proceedings of the National Academy of Sciences    of the United States of America, 87, 5061-5065.-   CLACKSON, T., ULTSCH, M. H., WELLS, J. A. & DE VOS, A. M. (1998)    Structural and functional analysis of the 1:1 growth    hormone:receptor complex reveals the molecular basis for receptor    affinity. Journal of Molecular Biology, 277, 1111-1128.-   CUNNINGHAM, B. C., ULTSCH, M., DE VOS, A. M., MULKERRIN, M. G.,    CLAUSER, K. R. & WELLS, J. A. (1991) Dimerization of the    extracellular domain of the human growth hormone receptor by a    single hormone molecule. Science, 254, 821-825.-   DE VOS, A. M., ULTSCH, M. & KOSSIAKOFF, A. A. (1992) Human growth    hormone and extracellular domain of its receptor: crystal structure    of the complex. Science, 255, 306-312.-   FUH, G., CUNNINGHAM, B. C., FUKUNAGA, R., NAGATA, S., GOEDDEL, D. V.    & WELLS, J. A. (1992) Rational design of potent antagonists to the    human growth hormone receptor. Science, 256, 1677-1680.-   HANIU, M., ARAKAWA, T., BURES, E. J., YOUNG, Y., HUI, J. O.,    ROHDE, M. F., WELCHER, A. A. & HORAN, T. (1998) Human leptin    receptor. Determination of disulfide structure and N-glycosylation    sites of the extracellular domain. Journal of Biological Chemistry,    273, 28691-28699.-   HIROIKE, T., HIGO, J., JINGAMI, H. & TOH, H. (2000) Homology    modeling of human leptin/leptin receptor complex. Biochemical &    Biophysical Research Communications, 275, 154-158.-   HUKSHORN, C. J., SARIS, W. H., WESTERTERP-PLANTENGA, M. S.,    FARID, A. R., SMITH, F. J. & CAMPFIELD, L. A. (2000) Weekly    subcutaneous pegylated recombinant native human leptin (PEG-OB)    administration in obese men. [see comments].Journal of Clinical    Endocrinology & Metabolism, 85, 4003-4009.-   KISHIMOTO, T., TAGA, T. & AKIRA, S. (1994) Cytokine signal    transduction. [Review] [92 refs]. Cell, 76, 253-262.-   LIVNAH, O., STURA, E. A., MIDDLETON, S. A., JOHNSON, D. L.,    JOLLIFFE, L. K. & WILSON, I. A. (1999) Crystallographic evidence for    preformed dimers of erythropoietin receptor before ligand    activation. Science, 283, 987-990.-   MAAMRA, M., FINIDORI, J., VON LAUE, S., SIMON, S., JUSTICE, S.,    WEBSTER, J., DOWER & ROSS, R. (1999) Studies with a growth hormone    antagonist and dual-fluorescent confocal microscopy demonstrate that    the full-length human growth hormone receptor, but not the truncated    isoform, is very rapidly internalized independent of Jak2-Stat5    signaling. Journal of Biological Chemistry, 274, 14791-14798.-   MELLADO, M., RODRIGUEZ-FRADE, J. M., KREMER, L., VON KOBBE, C., DE    ANA, A. M., MERIDA, I. & MARTINEZ, A. (1997) Conformational changes    required in the human growth hormone receptor for growth hormone    signaling. Journal of Biological Chemistry, 272, 9189-9196.-   ROSS, R. J., ESPOSITO, N., SHEN, X. Y., VON LAUE, S., CHEW, S. L.,    DOBSON, P. R., POSTEL-VINAY, M. C. & FINIDORI, J. (1997) A short    isoform of the human growth hormone receptor functions as a dominant    negative inhibitor of the full-length receptor and generates large    amounts of binding protein. Molecular Endocrinology, 11, 265-273.-   SUNDSTROM, M., LUNDQVIST, T., RODIN, J., GIEBEL, L. B., MILLIGAN, D.    & NORSTEDT, G. (1996) Crystal structure of an antagonist mutant of    human growth hormone, G120R, in complex with its receptor at 2.9 A    resolution. Journal of Biological Chemistry, 271, 32197-32203.-   SYED, R. S., REID, S. W., LI, C., CHEETHAM, J. C., AOKI, K. H., LIU,    B., ZHAN, H., OSSLUND, T D, CHIRINO, A. J., ZHANG, J., FINER-MOORE,    J., ELLIOTT, S., SITNEY, K., KATZ, B. A., MATTHEWS, D. J.,    WENDOLOSKI, J. J., EGRIE, J. & STROUD, R. M. (1998) Efficiency of    signalling through cytokine receptors depends critically on receptor    orientation. Nature, 395, 511-516.-   THORNER, M. O., STRASBURGER, C. J., WU, Z., STRAUME, M.,    BIDLINGMAIER, M., PEZZOLI, S., ZIB, K., SCARLETT, J. C. &    BENNETT, W. F. (1999) Growth hormone (GH) receptor blockade with a    PEG-modified GH (B2036-PEG) lowers serum insulin-like growth    factor-I but does not acutely stimulate serum GH. Journal of    Clinical Endocrinology & Metabolism, 84, 2098-2103.

1. A polypeptide comprising more than two ligand binding domains of acytokine receptor wherein said domains are linked by a linker moleculeand wherein the linker molecule comprises at least one proteolyticcleavage site.
 2. A polypeptide according to claim 1 wherein saidcleavage site is sensitive to a serum protease.
 3. A polypeptideaccording to claim 2 wherein the serum protease is thrombin.
 4. Apolypeptide according to claim 1 wherein said cleavage site comprisesthe amino acid sequence LVPRGS (SEQ ID: 1), or a variant thereof.
 5. Apolypeptide according to claim 1 wherein said cleavage site comprisesthe amino acid sequence SGGGG (SEQ ID:2), or a variant thereof.
 6. Apolypeptide according to claim 1 wherein said cleavage site comprisesthe amino acid sequence PGISGGGGGG (SEQ ID:3).
 7. A polypeptideaccording to claim 4 wherein said cleavage site comprises the amino acidsequence: LVPRGSPGISGGGGGG (SEQ ID:4), or a variant thereof.
 8. Apolypeptide according to claim 5 wherein said cleavage site comprises acenter and two copies of the amino acid sequence SGGGG, or a variantthereof, which flank the center of said cleavage site.
 9. A polypeptideaccording to claim 1 wherein said polypeptide comprises at least fourligand binding domains.
 10. A polypeptide according to claim 9 whereinsaid polypeptide comprises 4, 6, 8, 10, or 12 ligand binding domains.11. A polypeptide according to claim 1 wherein said polypeptidecomprises 3, 4, 5, 6, 7, 8, 9, or 10 ligand binding domains.
 12. Apolypeptide according to claim 9 wherein said polypeptide comprisesgreater than 10 ligand binding domains.
 13. A polypeptide according toclaim 1 wherein said polypeptide is an antagonist to said cytokine. 14.A polypeptide according to claim 1 wherein said polypeptide is anagonist to said cytokine.
 15. A polypeptide according to claim 1 whereinsaid cytokine receptor ligand binding domain is the ligand bindingdomain of a cytokine selected from the group consisting of: growthhormone; leptin; erythropoietin; prolactin; interleukins (IL), IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, the p35 subunit ofIL-12, IL-13, IL-15; granulocyte colony stimulating factor (G-CSF)granulocyte macrophage colony stimulating factor (GM-CSF); ciliaryneurotrophic factor (CNTF), cardiotrophin-1 (CT-1), leukaemia inhibitoryfactor (LIF), oncostatin M (OSM), interferon, IFNα and IFNγ.
 16. Apolypeptide according to claim 15 wherein the binding domain is theligand binding domain of growth hormone.
 17. A polypeptide according toclaim 16 wherein the binding domain is the ligand binding domain ofleptin.
 18. A polypeptide according to claim 1 wherein the linker is apolypeptide which comprises from 5 to 50 amino acid residues.
 19. Apolypeptide according to claim 18 wherein the linker comprises from 5 to30 amino acid residues.
 20. A polypeptide according to claim 1 whereinthe linker comprises at least one copy of the peptide GGGGS.
 21. Apolypeptide according to claim 20 wherein the linker is 5 amino acids inlength and consists of one copy of GGGGS (the Gly4Ser linker).
 22. Apolypeptide according to claim 20 wherein the linker is 10 amino acidsin length and consists of two copies of the Gly4Ser linker.
 23. Apolypeptide according to claim 20 wherein the linker is 15 amino acidsin length and consists of three copies of the Gly4Ser linker.
 24. Apolypeptide according to claim 20 wherein the linker is 20 amino acidsin length and consists of 4 copies of the Gly4Ser linker.
 25. Apolypeptide according to claim 1 wherein the polypeptide is a fusionprotein comprising inframe translational fusions of ligand bindingdomains.
 26. A polypeptide according to claim 1 comprising chemicalcrosslinkers wherein the chemical crosslinkers serve to link the ligandbinding domains.
 27. A polypeptide according to claim 26 wherein thechemical crosslinker comprises a homo-bifunctional crosslinker selectedfrom the group consisting ofdisuccinimidyl-suberimidate-dihydrochloride;dimethyl-adipimidate-dihydrochloride; and 1,5,-2,4 dinitrobenezene. 28.A polypeptide according to claim 26 or claim 27 wherein the crosslinkercomprises a hetero-bifunctional crosslinker selected from the groupconsisting of N-hydroxysuccinimidyl 2, 3-dibromopropionate;1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride; andsuccinimidyl 4-[n-maleimidomethyl]-cyclohexane-1-carboxylate.
 29. Anucleic acid molecule comprising a nucleic acid sequence which encodes apolypeptide according to claim
 1. 30. A nucleic acid molecule comprisingthe sequence selected from the group consisting of: (i) the sequencerepresented by FIG. 4 or 6; (ii) a sequence which hybridises to thesequence of (i) above and which has cytokine receptor modulatingactivity; and (iii) a sequence which is degenerate as a result of thegenetic code to the sequences defined in (i) and (ii) above.
 31. Anucleic acid molecule which hybridises under stringent hybridisationconditions to the sequences represented in FIG. 4 or
 6. 32. Apolypeptide encoded by the nucleic acid molecule according to claim 29.33. A polypeptide according to claim 32 wherein said polypeptide ismodified by deletion, addition, and/or substitution of at least oneamino acid residue and said modification enhances the antagonistic oragonistic effects of said polypeptide with respect to the inhibition oractivation of receptor mediated cell signalling.
 34. A vector comprisingthe nucleic acid molecule of claim
 29. 35. A vector according to claim34 wherein said vector is an expression vector adapted for prokaryoticor eukaryotic gene expression.
 36. A vector according to claim 34wherein said vector further encodes, a secretion signal linked to thepolypeptide to facilitate purification of the polypeptide.
 37. A methodto prepare a polypeptide according to claim 1, the method comprising;(i) growing a cell transformed or transfected with a nucleic acid ofclaim 29 in conditions conducive to the manufacture of said polypeptide;and (ii) purifying said polypeptide from said cell, or its growthenvironment.
 38. A cell transformed/transfected with the vector of thenucleic acid of claim
 29. 39-42. (canceled)
 43. A pharmaceuticalcomposition comprising the polypeptide according to claim 1, and apharmaceutically acceptable carrier, excipient, or a diluent.
 44. Apharmaceutical composition comprising the nucleic acid molecule of claim29 and a pharmaceutically acceptable excipient.
 45. A method fortreating a disease selected from the group consisting of: acromegaly;gigantism; GH deficiency; Turners syndrome; renal failure; osteoporosis;diabetes mellitus; cancer; obesity; insulin resistance; hyperlipidaemia;hypertension; anaemia; an autoimmune disease; an infectious disease; aninflammatory disorder, and rheumatoid arthritis, wherein said methodcomprising administering to a patient in need thereof a pharmaceuticalcomposition according to claim
 43. 46. A method for treating a diseaseselected from the group consisting of: acromegaly; gigantism; GHdeficiency; Turners syndrome; renal failure; osteoporosis; diabetesmellitus; cancer; obesity; insulin resistance; hyperlipidaemia;hypertension; anaemia; an autoimmune disease; an infectious disease; aninflammatory disorder, and rheumatoid arthritis, wherein said methodcomprising administering to a patient in need thereof a pharmaceuticalcomposition according to claim 44.